Method of imparting immunomodulating and antiviral activity

ABSTRACT

Compounds of the formula ##STR1## where X is OH, NH 2 , SH, OR or SR (where R is alkyl of 1 to 4 carbon atoms or benzyl), R 1  is H or alkyl of 1 to 8 carbon atoms, R 2  is H or methyl, Y is the salt of an amine of the formula ##STR2## where R 3  and R 4  are lower alkyl, e.g., 1 to 4 carbon atoms and n is an integer of 2 to 4 with p-acetamidobenzoic acid and where z is a number from 0 to 10 are useful as immunomodulators, as antiviral agents and in specific cases have anti-leukemic activity. The compounds and compositions where z is 1 to 10 are novel per se. When R 2  is H the presence of Y enhances the immunoregulatory activity and the antiviral activity. If X is the NH 2  there is immunoinhibitory activity but no immunostimulatory (immunopotentiatory) activity.

This is a division of application Ser. No. 942,802, filed Sept. 15,1978.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that compounds of theformula ##STR3## where X is OH, NH₂, SH, OR or SR where R is alkyl of 1to 4 carbon atoms or benzyl, R¹ is H or alkyl of 1 to 8 carbon atoms, R²is H or methyl, Y is the salt of an amine of the formula ##STR4## whereR³ and R⁴ are lower alkyl, e.g., 1 to 4 carbon atoms and n is an integerof 2 to 4 with p-acetamidobenzoic acid and where z is a number from 0 to10 are useful as immunomodulators, as antiviral agents and in specificcases have anti-leukemic activity. The compounds and compositions wherez is 1 to 10 are novel per se. When R² is H the presence of Y enhancesthe immunoregulatory activity and the antiviral activity. If X is theNH₂ there is immunoinhibitory activity but no immunostimulatory(immunopotentiatory) activity.

Immunoregulatory activity appears to increase with increasing chainlength for R¹, at least from methyl through hexyl. Preferably R¹ isn-alkyl, i.e., methyl, ethyl, n-propyl, n-butyl, n-amyl, n-hexyl,n-heptyl or n-octyl. R² is preferably methyl. R can be methyl, ethyl,n-propyl, n-butyl, isopropyl, etc. When X is NH₂ the compound can bepresent as the free base or as the salt with a non-toxic acid, i.e.,pharmaceutically acceptable acid, e.g., hydrochloric acid, hydrobromicacid, sulfuric acid, phosphonic acid, citric acid, lactic acids,tartaric acid, salicylic acid, acetyl salicyclic acid, acetic acid,propionic acid, p-toluene sulfonic acid, methane sulfonic acid, maleicacid, succinic acid, malonic acid, adipic acid.

A preferred class of amines to form the salt with para acetamidobenzoicacid has the formula ##STR5## where R⁵ and R⁶ are lower alkyl, e.g.,methyl, ethyl, propyl, isopropyl or butyl and n is an integer of 2 to 4.Typical examples of such amines include dimethylamino ethanol,dimethylamino isopropanol, diethylamino ethanol, diethylaminoisobutanol, diethylamino isopropanol, methyl ethyl amino ethanol,diisobutylamino-N-butanol, dimethylamino propanol,dimethylamino-N-butanol, diisobutylamino ethanol, dimethylamino butanol,dibutylamino-N-butanol, dibutylamino ethanol, dipropylamino ethanol anddiisopropylamino ethanol. The presently preferred amine is dimethylaminoisopropanol. When Y is present, i.e., z is 1 to 10, preferably z is 3.However, z can also be 1, 2, 4, 5, 6, 7, 8, 9 or 10.

While there are preferably used the compounds where Y is the salt of theamine ##STR6## with p-acetamidobenzoic acid there can also be used saltsof the formula Y¹ wherein the amine is as just defined the acid is apharmaceutically acceptable acid other than p-acetamidobenzoic acid,e.g., hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoricacid, acetic acid, propionic acid, malonic acid, lactic acid, citricacid, tartaric acid, p-toluene sulfonic acid, adipic acid, maleic acid,succinic acid, methane sulfonic acid, salicylic acid, acetyl salicylicacid.

In describing the compounds below, when Y is present the abbreviationDIP.PAcBA stands for dimethylamino-2-propanol-p-acetamido benzoate.Unless a number in parentheses, e.g. (10), follows this abbreviation,then Y is 3. If a number in parentheses follows the abbreviationDIP.PAcBA there the number indicates the number of moles of Y groupspresent to 1 mole of the 9-(hydroxyalkyl)purine.

In Table 1 below the compounds are believed to be pure except forcompound 15443 which is believed to also contain a salt in addition tothe compound of the invention.

An immunomodulator is a compound which regulates the immune response.Thus it covers both immunostimulation (immunopotentiation) andimmunoinhibition. Immunostimulation, of course, is useful in building upimmunity. Immunoinhibition also has utility in a number of areas. Forexample, it is useful in organ transplants, e.g., kidney or hearttransplants, to prevent rejection.

In the tables showing the immunopotentiating properties of thecompounds, a plus or a minus (-) indicates immunostimulating orimmunoinhibiting properties. Respectively the number 0 indicates thecompound had neither immunopotentiating activity of immunoinhibitingactivity.

There are included in some of the tables several compounds wherein X isnot within that claimed. These non-claimed compounds as a rule haverelatively low activities and are included to illustrate the fact thatthe X group can have a significant effect on the properties of thecompounds.

A mitogen is a substance which induces cell proliferation, as occursduring immunization.

Table 1 (excluding compounds 15427 and 15423) shows compounds useful inthe invention.

The synthetic procedures A through L mentioned in Table 1 are describedin more detail subsequently.

The compositions of the invention are useful in treating mammals (andcells of mammals) including humans, swine, dogs, cats, cattle, horses,sheep, goats, mice, rabbits, rats, guinea pigs, hamsters, monkeys, etc.

Unless otherwise indicated, all parts and percentages are by weight.

All temperatures are in degrees centigrade unless otherwise indicated.

The compositions can comprise, consist essentially of or consist of thematerials set forth and the processes can comprise, consist essentiallyof or consist of the steps set forth with such materials.

The compositions can be administered to the mammals by conventionaltechniques, e.g., orally, nasally, rectally, vaginally, enterally orparenterally. They can be employed as injectable solutions, e.g., inwater, or as tablets, pills, capsules, etc.

                                      Table 1                                     __________________________________________________________________________    SUMMARY OF CHEMICAL PROPERTIES OF 9-(HYDROXYALKYL) PURINES                     ##STR7##                                                                                                UV Spectra              Elemental                  Compound              Synthetic          Con       Analysis                   No. R.sup.1                                                                           R.sup.2                                                                          X  Y       Method                                                                             M.Pt. °C.                                                                    λMax.                                                                      λMin.                                                                      10.sup.-3                                                                          pH   C    H   N                 __________________________________________________________________________    15425                                                                             H   H  OH --      D    274°                                                                         250  222.5                                                                            11.93                                                                              7                                                                250 219 11.0 1                                                                254  221.5                                                                            12.53                                                                              10                              15428                                                                             H   H  OH DIP . PAcBA                                                                           L                                                                                        323 251 23.0 7                               15435                                                                             H   H  SH --      C    278-80                                                                              323 252 19.9 1                                                                323 251 19.9 10                              15437                                                                             H   H  SH DIP . PAcBA                                                                           L                                                                                        250  223.5                                                                            11.0 7                               15446                                                                             H   CH.sub.3                                                                         OH --      A    244-5 250 220 10.6 1                                                                254  223.5                                                                            12.1 10                              15447                                                                             H   CH.sub.3                                                                         OH DIP . PAcBA                                                                           L                                                                                        261 228 15.8 7    Cal 49.73                                                                          5.74                                                                              36.25             15431                                                                             H   CH.sub.3                                                                         NH.sub.2                                                                         --      B    188°                                                                         259 231 15.4 1    FD 49.56                                                                           5.62                                                                              36.22                                              261 225 15.7 10                              15432                                                                             H   CH.sub.3                                                                         NH.sub.2                                                                         DIP . PAcBA                                                                           L                                                       15427                                                                             CH.sub.3                                                                          H  I  --      E    178°                                                                         276 237 10.9 7    Cal 31.60                                                                          2.98                                                                              18.43                                              276 237 10.9 1    FD31.53                                                                            2.96                                                                              18.18                                              276 237 10.9 10                                                               265 228 9.1  7    Cal 45.20                                                                          4.26                                                                              26.36             15423                                                                             CH.sub.3                                                                          H  Cl --      F    200-204                                                                             265 228 9.1  1    FD 45.11                                                                           4.27                                                                              26.25                                              265 228 9.1  10                                                                261.5                                                                            228 13.56                                                                              7                               15433                                                                             CH.sub.3                                                                          H  NH.sub.2                                                                         --      G    215-16                                                                              259 231 13.26                                                                              1                                                                261  224.5                                                                            13.80                                                                              10                              15434                                                                             CH.sub.3                                                                          H  NH.sub.2                                                                         DIP . PAcBA                                                                           L                                                                                        250 223 7.52 7                               15443                                                                             CH.sub.3                                                                          H  OH --      H    198-199                                                                             250 218 6.91 1                                                                255  225.5                                                                            7.91 10                              15444                                                                             CH.sub.3                                                                          H  OH DIP . PAcBA                                                                           L                                                                                        250 224 11.09                                                                              7    Cal 59.07                                                                          7.65                                                                              21.16             15417                                                                             C.sub.6 H.sub.13                                                                  H  OH --      I    226° C.                                                                      250 220 10.37                                                                              1    FD 59.01                                                                           7.55                                                                              21.24                                              255 223 11.96                                                                              10                              15418                                                                             C.sub.6 H.sub.13                                                                  H  OH DIP . PAcBA                                                                           L                                                                                        250 224 12.1 7    Cal 60.41                                                                          7.97                                                                              20.13             15392                                                                             C.sub.6 H.sub.13                                                                  CH.sub.3                                                                         OH --      J    202° C.                                                                      248 222 13.3 1    FD 60.47                                                                           7.86                                                                              20.08                                              254 220 14.1 10                              15410                                                                             C.sub.6 H.sub.13                                                                  CH.sub.3                                                                         OH DIP . PAcBA                                                                           L                                                                                        261 230 9.77 7    Cal 58.58                                                                          7.71                                                                              22.32             15426                                                                             C.sub.6 H.sub.13                                                                  CH.sub.3                                                                         NH.sub.2                                                                         HCl Salt                                                                              K    176°-9° C.                                                            259 233 9.60 1    FD 53.56                                                                           7.67                                                                              22.24                                              261 235 9.77 10                              __________________________________________________________________________

Other compounds within the invention are set forth in Table 1a belowwherein the basic formula is the same as that in Table 1. In Tables 1and 1a, the alkyl groups for R¹ are all n-alkyl.

                  Table 1a                                                        ______________________________________                                        COMPOUND                                                                      R.sup.1  R.sup.2  X           Y                                               ______________________________________                                        C.sub.6 H.sub.13                                                                       CH.sub.3 OH          DIP . PAcBA(10)                                 C.sub.6 H.sub.13                                                                       CH.sub.3 OH          DIP . PAcBA(1)                                  H        CH.sub.3 OH          DIP . PAcBA(10)                                 H        CH.sub.3 OH          DIP . PAcBA(1)                                  CH.sub.3 CH.sub.3 OH           --                                             CH.sub.3 CH.sub.3 OH          DIP . PAcBA                                     C.sub.2 H.sub.5                                                                        H        OH          DIP . PAcBA                                     C.sub.2 H.sub.5                                                                        H        OH           --                                             C.sub.3 H.sub.7                                                                        H        OH           --                                             C.sub.3 H.sub.7                                                                        H        OH          DIP . PAcBA                                     C.sub.2 H.sub.5                                                                        CH.sub.3 OH           --                                             C.sub.2 H.sub.5                                                                        CH.sub.3 OH          DIP . PAcBA                                     C.sub.2 H.sub.7                                                                        CH.sub.3 OH           --                                             C.sub.3 H.sub.7                                                                        CH.sub.3 OH          DIP . PAcBA                                     C.sub.4 H.sub.9                                                                        H        OH           --                                             C.sub.4 H.sub.9                                                                        H        OH          DIP . PAcBA                                     C.sub.4 H.sub.9                                                                        CH.sub.3 OH           --                                             C.sub.4 H.sub.9                                                                        CH.sub.3 OH          DIP . PAcBA                                     C.sub.5 H.sub.11                                                                       H        OH           --                                             C.sub.5 H.sub.11                                                                       H        OH          DIP . PAcBA                                     C.sub.5 H.sub.11                                                                       CH.sub. 3                                                                              OH          DIP . PAcBA                                     C.sub.5 H.sub.11                                                                       CH.sub.3 OH           --                                             C.sub.7 H.sub.15                                                                       H        OH           --                                             C.sub.7 H.sub.15                                                                       H        OH          DIP . PAcBA                                     C.sub.7 H.sub.15                                                                       CH.sub.3 OH           --                                             C.sub.7 H.sub.15                                                                       CH.sub.3 OH          DIP . PAcBA                                     C.sub.8 H.sub.17                                                                       H        OH           --                                             C.sub.8 H.sub.17                                                                       H        OH          DIP . PAcBA                                     C.sub.8 H.sub.17                                                                       CH.sub.3 OH           --                                             C.sub.8 H.sub.17                                                                       CH.sub.3 OH          DIP . PAcBA                                     C.sub.6 H.sub.13                                                                       CH.sub.3 OCH.sub.3    --                                             C.sub.6 H.sub.13                                                                       CH.sub.3 OCH.sub.3   DIP . PAcBA                                     C.sub.6 H.sub.13                                                                       H        OCH.sub.3   DIP. PAcBA                                      C.sub.6 H.sub.13                                                                       H        OCH.sub.3    --                                             CH.sub.3 H        OCH.sub.3    --                                             CH.sub.3 H        OCH.sub.3   DIP . PAcBA                                     H        H        OCH.sub.3    --                                             H        H        OCH.sub.3   DIP . PacBA                                     H        CH.sub.3 OCH.sub.3   DIP . PAcBA                                     H        CH.sub.3 OCH.sub.3    --                                             C.sub.6 H.sub.13                                                                       CH.sub.3 OC.sub.2 H.sub.5                                                                           --                                             C.sub.6 H.sub.13                                                                       CH.sub.3 OC.sub.2 H.sub.5                                                                          DIP . PAcBA                                     C.sub.6 H.sub.13                                                                       H        OC.sub.2 H.sub.5                                                                          DIP . PAcBA                                     C.sub.6 H.sub.13                                                                       H        OC.sub.2 H.sub.5                                                                           --                                             C.sub.6 H.sub.13                                                                       CH.sub.3 OC.sub.3 H.sub.7                                                                           --                                             C.sub.6 H.sub.13                                                                       CH.sub.3 OC.sub.3 H.sub.7                                                                          DIP . PAcBA                                     CH.sub.3 H        OC.sub.3 H.sub.7                                                                          DIP . PAcBA                                     CH.sub.3 H        OC.sub.3 H.sub.7                                                                           --                                             H        H        OC.sub.3 H.sub.7                                                                          DIP . PAcBA                                     H        CH.sub.3 OC.sub.3 H.sub.7                                                                          DIP . PAcBA                                     C.sub.6 H.sub.13                                                                       CH.sub.3 OC.sub.4 H.sub.9                                                                           --                                             C.sub.6 H.sub.13                                                                       CH.sub.3 OC.sub.4 H.sub.9                                                                          DIP . PAcBA                                     C.sub.6 H.sub.13                                                                       H        OC.sub.4 H.sub.9                                                                           --                                             C.sub.6 H.sub.13                                                                       H        OC.sub.4 H.sub.9                                                                          DIP . PAcBA                                     H        H        OC.sub.4 H.sub.9                                                                           --                                             H        H        OC.sub.4 H.sub.9                                                                          DIP . PAcBA                                     H        CH.sub.3 OC.sub.4 H.sub.9                                                                           --                                             H        CH.sub.3 OC.sub.4 H.sub.9                                                                          DIP . PAcBA                                     CH.sub.3 CH.sub.3 OC.sub.4 H.sub.9                                                                           --                                             CH.sub.3 CH.sub.3 OC.sub.4 H.sub.9                                                                          DIP . PAcBA                                     CH.sub.3 H        OC.sub.4 H.sub.9                                                                           --                                             CH.sub.3 H        OC.sub.4 H.sub.9                                                                          DIP . PAcBA                                     C.sub.6 H.sub.13                                                                       CH.sub.3 SCH.sub.3    --                                             C.sub.6 H.sub.13                                                                       CH.sub.3 SCH.sub.3   DIP . PAcBA                                     C.sub.6 H.sub.13                                                                       H        SCH.sub.3    --                                             C.sub.6 H.sub.13                                                                       H        SCH.sub.3   DIP . PAcBA                                     CH.sub.3 CH.sub.3 SCH.sub.3    --                                             CH.sub.3 CH.sub.3 SCH.sub.3   DIP . PAcBA                                     CH.sub.3 H        SCH.sub.3    --                                             CH.sub.3 H        SCH.sub.3   DIP . PAcBA                                     H        H        SCH.sub.3    --                                             H        H        SCH.sub.3   DIP . PAcBA                                     H        CH.sub.3 SCH.sub.3   DIP . PAcBA                                     H        CH.sub.3 SCH.sub.3    --                                             C.sub.6 H.sub.13                                                                       CH.sub.3 SC.sub.4 H.sub. ----                                        C.sub.6 H.sub.13                                                                       CH.sub.3 SC.sub.4 H.sub.9                                                                          DIP . PAcBA                                     C.sub.6 H.sub.13                                                                       H        SC.sub.4 H.sub.9                                                                          DIP . PAcBA                                     C.sub.6 H.sub.13                                                                       H        SC.sub.4 H.sub.9                                                                           --                                             CH.sub.3 H        SC.sub.4 H.sub.9                                                                           --                                             CH.sub.3 H        SC.sub.4 H.sub.9                                                                          DIP . PAcBA                                     H        H        SC.sub.4 H.sub.9                                                                           --                                             H        H        SC.sub.4 H.sub.9                                                                          DIP . PAcBA                                     H        CH.sub.3 SC.sub.4 H.sub.9                                                                          DIP . PAcBA                                     H        CH.sub.3 OH          DIP . PAcBA(10)                                 H        CH.sub.3 OH          DIP . PAcBA(1)                                  C.sub.6 H.sub.13                                                                       H        O-benzyl     --                                             C.sub.6 H.sub.13                                                                       H        O-benzyl    DIP . PAcBA                                     C.sub.6 H.sub.13                                                                       CH.sub.3 O-benzyl     --                                             C.sub.6 H.sub.13                                                                       CH.sub.3 O-benzyl    DIP . PAcBA                                     C.sub.6 H.sub.13                                                                       CH.sub.3 S-benzyl     --                                             C.sub.6 H.sub.13                                                                       CH.sub.3 S-benzyl    DIP . PAcBA                                     C.sub.6 H.sub.13                                                                       H        S-benzyl     --                                             C.sub.6 H.sub.13                                                                       H        S-benzyl    DIP . PAcBA                                     ______________________________________                                    

DESCRIPTION OF THE PREFERRED EMBODIMENTS Method A9-(2-HYDROXY-1-PROPYL)HYPOXANTHINE (NPT 15446) ##STR8##

9-2-Hydroxy-1-propyl)adenine (I, 4.0 g, 20.7 mmol) was suspened in 50%acetic acid (20 ml) and sodium nitrite (4 g, 58 mmol), was slowly added.The mixture was stirred at 25° for 3 hr. The resulting solution wasevaporated to dryness and isopropanol added; this operation was repeatedonce. The solid residue was boiled in isopropanol and filtered. Thefiltrate was evaporated and crystallized by addition of acetone.Recrystallization was made from iso-propanol/methanol (98:2); acolorless crystalline product was obtained. Yield 3.3 g (82%) M.P.244°-250° uv (H₂ O; pH 5.5) λmax 250 nm

Method B 9-(2-HYDROXY-1-PROPYL)-6-CHLOROPURINE ##STR9##

There were employed the methods of Schaeffer, H. J., Vogel, D. andVince, R., J. Med. Chem. 8,502 (1965); and Schaeffer, H. J. and Vince,R., J. Med. Chem. 10, 689 (1967).

A solution of 5-amino-4,6-dichloropyrimidine (I, 20 g, 0.12 mole) in 11%ethanolic solution of isopropanolamine (200 ml) was refluxed for 8 hr.The reaction mixture was evaporated to a syrup, ethanol added andevaporated again; this operation was repeated once. The resulting syrupwas poured into water (300 ml) giving a crystalline mass. It wascollected by filtration, washed with water and dried to give 19 g ofcrude 9-(2-hydroxy-1-propylamino)5-amino-6 chloropyrimidine (II).

The crude compound II was suspended in triethylorthoformate (120 ml) towhich ethanesulfonic acid (5 drops) was added. After 15 min. all thesolid dissolved and the solution was kept at 25° overnight. Evaporationin vacuo gave a thick syrup which was submitted to high vacuoevaporation to remove the excess of isopropanolamine. Uponcrystallization with xylene, 5 g of crude material was obtained.

Method B 9-(2-HYDROXY-1-PROPYL)ADENINE (NPT 15431) ##STR10##

9-(2-Hydroxy-1-propyl)-6-chloropurine (I, 9 g, 42.5 mmol) was dissolvedin saturated methanolic ammonia and ammonium chloride (50 mg). Themixture was heated at 130° in a bomb for 6 hr. The resulting solutionwas evaporated to dryness and recrystallized from ethanol/acetone.Yield=6.68 g of a colorless crystalline product (81%) mp 193°-194° uv(H₂ O; pH 5.5) λmax 260 nm TLC in CHl₃ :MeOH (5:1) R_(f) 0.44

Anal. Calc. for C₈ H₁₁ N₅ O: C, 49.73; H, 5.74; N, 36.25; Found: C,49.56, H, 5.62; N, 36.22.

Method C 9-(1-HYDROXYETHYL)-6-MERCAPTOPURINE (NPT 15435) ##STR11##

There was employed the method of Schaeffer and Bhargava, Biochemistry 4,71 (1965).

9-(1-Hydroxyethyl)-6-chloropurine (I, 2 g, 0.01 mol) and thiourea (0.76g; 0.01 mol) were dissolved in ethanol (15 ml) and refluxed for 30 min.The resulting precipitate was collected by filtration and suspended inwater to form a slurry. Neutralization with sodium acetate gavecolorless crystals. Yield 1.5 g (76%).

M.P. 278°-280°; uv (H₂ O, pH 5.5) λmax 320, 230 nm.

Method D 9-HYDROXYETHYL HYPOXANTHINE (NPT 15425) ##STR12##

There was used the method of Schaeffer, H. J. and Bhargava, P. S.,Biochemistry 4, 71 (1965).

6-Chloro-9-hydroxyethyl purine, III (4 g), was added slowly to warm NNaOH (30 ml) and refluxed for 2 hr. The reaction is cooled in ice andneutralized with glacial acetic acid. After filtration, portions ofunreacted III are removed. The product is recrystallized from methanoland washed with acetone. Colorless crystals. Yield, 1 g. (28%); mp.274°; uv (H₂ O, pH 5.5), λmax 250 nm.

Method E 9-(1-HYDROXYL-2-PROPYL)-6-IODOPURINE (NPT 15427) ##STR13##

9-(1-Hydroxy-2-propyl)-6-chloropurine (I, 1.5 g, 7 mmol) was added tohydroiodic acid (15 ml) at -10° with stirring for 45 min. Theprecipitate was filtered, neutralized with anhydrous sodium acetate at5°, and washed with a little cold water (3 times). Recrystallizationfrom ethanol/H₂ O, gave colorless crystals. Yield=0.9 g (42%)mp=193°-194° uv λmax 276 nm (H₂ O, pH 5.5).

Anal. Calc. for C₈ H₉ N₄ OI MW=304.1: C, 31.60; H, 2.98; N, 18.43; I,41.73. Found: C, 31.53; H, 2.96; N, 18.18; I, 41.70.

Method F 9-(1-HYDROXY-2-PROPANE)-6-CHLOROPURINE (NPT 15423) ##STR14##

There was used the method of Schaeffer, H. J. and Schwender, C. F., J.Med. Chem. 17, 6 (1974).

A solution of 5-amino-4,6-dichloropyrimidine (I, 6.56 g 40 mmol) and2-amino-1-propanol (II, 3.3 g, 44 mmol) was refluxed in n-pentanol (288ml) and tert-butylamine (96 ml) for 45 hr. under N₂ atmosphere. Thesolution was evaporated to a syrup and ethanol added 4 times andevaporated. The resulting syrup was suspended in triethylorthoformate(150 ml) and ethanesulfonic acid (10 drops). The suspension wasvigorously stirred overnight, then evaporated to dryness, ethanol addedand this operation repeated three times. Crystallization of colorlessproduct occurs during evaporation. The crystals were filtered, and thefiltrate was evaporated, ethanol added and this operation repeated threetimes to give a crude material (3.6 g).

Recrystallized from 98% aqueous ethanol. uv (H₂ O, pH 5.5) λmax 265 nm;mp 201°-203°; yield 2.79 (32%).

Anal. C₈ H₉ N₄ OCl. Calc. C, 45.20; H, 4.26; N, 26.36; Cl, 16.68. Found:C, 45.11; H, 4.27; N, 26.25; Cl, 16.71.

Method G 9-(1-HYDROXY-2-PROPYL)ADENINE (NPT 15433) ##STR15##

There was used the procedure of Schaeffer, H. and Schwender, C., J.Pharm. Sci., 60, 1204 (1971). Also Schaeffer et al., J. Med. Chem. 15,456 (1972).

9-(1-Hydroxy-2-propyl)-6-chloropurine (I, 2.0 g, 9.4 mmol) was suspendedin methanol/ammonia (30 ml) and ammonium chloride (50 mg) added as acatalyst and the mixture heated at 130° for 4.5 hr; the solution wasevaporated to dryness. Recrystallization from ethanol of the obtainedcrude product gave colorless needles. Yield=1.15 g (63%) mp=215°-216° uv(H₂ O, pH 5.5) λmax, 260 nm.

Method H 9-(1-HYDROXY-2-PROPYL)HYPOXANTHINE (NPT 15443) ##STR16##

9-(1-Hydroxy-2-propyl)adenine (I, 4 g, 21 mmol) was dissolved in 50%acetic acid (20 ml), sodium nitrite (4 g, 58 mmol) added and the mixturestirred at 25° for 31/2 hr. The solution was evaporated to dryness twicewith isopropanol. The residue was taken up in isopropanol and filtered,the precipitate discarded, and the filtrate evaporated to form a gelwhich, upon the addition of acetone, solidified. Yield=3.65 (90%) ofcolorless crystals. Recrystallized from isopropanol/methanol (98:2).mp=202°-207° TLC in CHCl₃ :MeOH (5:1) 1 spot R_(f) --0.30 uv (H₂ O, pH5.5)=λmax 250 nm.

Method I COMPOUND NPT 15417

There was used the procedure of Schaeffer et al, Journal ofPharmaceutical Sciences 16:1204-1210, Method F.

The product is compound XL in Table III of Schaeffer et al.

Method J ERYTHRO-9-(2-HYDROXY-3-NONYL)HYPOXANTHINE (NPT 15392)

An outline of the synthetic sequence for the preparation oferythro-9-(2-hydroxy-3-nonyl)hypoxanthine (Nonylhypoxanthine, VIII) isshown in Flow Charts 1 and 2. The improvements over the procedure of H.J. Schaeffer and C. F. Schwender, J. Med. Chem., 17, 6 (1974) in thereaction sequence leading to theerythro-9-(2-hydroxy-3-nonyl)-6-chloropurine (VII) are indicated. Thelast step, the hydrolysis of the 6-chloropurine derivative (VII), toyield nonylhypoxanthine (VIII) is an adaptation of the method reportedby A. Giner-Sorolla, C. Gryte, A. Bendich and G. B. Brown, J. Org. Chem.34, 2157 (1969) for the hydrolysis of halogenopurines.

The alternate route, i.e., the nitrosation oferythro-9-(2-hydroxy-3-nonyl)adenin (EHNA) (IX), to yieldNonylhypoxanthine (VIII) (shown on Flow Chart 2) consists of theprevious conversion by ammonolysis of the chloro derivative (VII) intothe aminopurine (IX, EHNA) followed by its nitrosation to yieldNonylhypoxanthine (VIII).

Flow Chart 1 OUTLINE OF THE SYNTHESIS OFERYTHRO-9-(2-HYDROXY-3-NONYL)HYPOXANTHINE (VIII) Step 1ACETAMIDONONAN-2-ONE (II)

Acylation of 2-amino octanoic acid ##STR17##

Step 2 ACETAMIDONONAN-2-ONE HYDROCHLORIDE (III)

Formation of the acetamidononan-2-one hydrochloride ##STR18##

Step 3 ERYTHRO-3-AMINO-2-NONANOL (IV)

Reduction of the acetamidononan-2-one hydrochloride ##STR19##

(Figures below the arrow refer to % yield.)

Step 4 ERYTHRO-5-AMINO-4-CHLORO-6-(2-HYDROXY-3-NONYLAMINO)PYRIMIDINE(VI)

Condensation of erythro-3-amino-2-nonanol with 5amino-4,6-dichloropyrimidine ##STR20##

Step 5 ERYTHRO-9-(2-HYDROXY-3-NONYL)-6-CHLOROPURINE (VII)

Ring closure oferythro-5-amino-4-chloro-6-(2-hydroxy-3-nonylamino)pyrimidine (V)##STR21##

Step 6 ERYTHRO-9-(2-HYDROXY-3-NONYL)HYPOXANTHINE (VIII)

(By hydrolysis of the 6-chloropurine derivative) ##STR22##

Flow Chart 2 ALTERNATIVE ROUTE FOR THE PREPARATION OFERYTHRO-9-(2-HYDROXY-3-NONYL HYPOXANTHINE (VIII) Step 1aERYTHRO-9-(2-HYDROXY-3-NONYL)ADENINE (IX)

Ammonolysis of erythro-9-(2-hydroxy-3-nonyl)-6-chloropurine (VII)##STR23##

Step 2b ERYTHRO-9-(2-HYDROXY-3-NONYL)HYPOXANTHINE (VIII)

Nitrosation of erythro-9-(2-hydroxy-3-nonyl)adenine (IX) ##STR24##

3-ACETAMIDONONAN-2-ONE (II) ##STR25##

A mixture of 2-amino-1-octanoic acid (I, 200 g, 1.26 mole) in aceticanhydride (960 ml), and pyridine (640 ml) was heated on a boiling waterbath for 4 hr. The reaction mixture was evaporated in vacuo, and theresidue was partitioned 6-8 times between 5% aqueous solution of NaHCO₃(400 ml) and ether (400 ml). The combined ethereal extracts were driedwith anhydrous MgSO₄ and evaporated to dryness to give crude3-acetamidononan-2-one, 154 g (70%).

3-AMINO-2-NONANONE HYDROCHLORIDE (III) ##STR26##

The crude product (II) obtained in the preceding operation (154 g) wasdissolved in concentrated aqueous HCl (1,540 ml) and refluxed for 2 hr.and then evaporated to dryness in vacuo. The resulting solid wasrecrystallized from a warm solution in EtOH (200 ml) and then cooled to25°. To this solution ether (600 ml) was added. A white crystallineprecipitate appears; the suspension is kept at 5° overnight. Theprecipitate is collected and washed with ether (once with 100 ml) togive 125 g (67%) white crystalline product M.P. 112° dec.

If the crystalline material were not white or had a lower melting point,it should be recrystallized with charcoal from tetrahydrofuran. In onerepeat of this procedure there was used 150 ml of hydrofuran for 100 gof the crude hydrochloride (III).

ERYTHRO-3-AMINO-2-NONANOL (IV) ##STR27##

3-Amino-2-nonanol hydrochloride (43.8 g, 0.226 mole) was dissolved inabsolute methanol (150 ml) and cooled to -10° in an ice-salt bath. 1/Potassium borohydride (24.4 g, 0.45 mole) 2/ was added in small portionsover a 2-3 hr. period. The mixture is then kept at -10° to -15° for 3hr. 3,4/ and slowly allowed to reach room temperature (22°), thenstirred overnight (20 hr.) at room temperature. The mixture is thenevaporated to dryness (syrup) in vacuo and partitioned between H₂ O (150ml) and chloroform (150 ml). The H₂ O layer was further extracted (3x)with chloroform (100 ml ea.). The chloroform layer was dried with MgSO₄and evaporated in vacuo to give a slightly yellowish, oily product. Thisliquid was distilled in high vacuo at 95°-100° (0.15 mm Hg) to give pureerythro-3 -amino-2-nonanol, 26.4 g, 75% yield, m.p. 81°-86°.

1. Upon cooling the solution of III, some material precipitates; thishas no effect on the outcome of the reaction.

2. At this point, the present procedure differs from that of Schaefferet al. Schaeffer adds acetic acid at the same time as KBH₄, maintainingthe pH at 5-6. It has been found that neutralization entails loss ofKBH₄ and that a pH above 5 is tolerated. More important is the fact thatthe simultaneous addition of acetic acid and KBH₄ (as proposed bySchaeffer) makes the reaction very difficult to control. The temperatureraises considerably and losses in yield and/or quality of the productoccur.

3. It is recommended to use an efficient stirring to insure the properreaction which will be completed when all the small lumps and portionsof potassium borohydride have disappeared.

4. Cooling at 0°, as described by Schaeffer et al (Method D, line 4 andff.) is insufficient. It is an improvement to keep the reaction wellbelow 0°; it is best to keep it below -10° all the time. If thetemperature is allowed to go over -10°, substantial loss in yield mayresult.

ERYTHRO-5-AMINO-4-CHLORO-6-(2-HYDROXY-3-NONYLAMINO)PYRIMIDINE (VI)##STR28##

A mixture of 4,6-dichloro-5-aminopyrimidine (V, 24.6, 0.15 mole) anderythro-3-amino-2-nonanol (IV, 26.2 g, 0.164 mole) in 1-pentanol (1.080ml) and tributylamine (350 ml) was prepared with stirring at 25°. Theresulting suspension was heated to reflux under nitrogen atmosphere for28 hr. (solution took place in about 1/2 hr.). At that time a sample ofthe reaction product showed a uv λmax 267 and 297 nm (H₂ O, pH 5.5).

The resulting solution was concentrated in a hot water bath at 10 mmpressure to a syrup and further evaporated in an oil bath at 0.1 mm and100° to yield a viscous liquid to which n-hexane (450 ml) was added. Themixture was refluxed for 1 hr., and the hot, yellowish hexanesupernatant was separated from the liquid at the bottom of the roundbottom flask.

The resulting light brown oil from which any residual hexane wasevaporated in vacuo and dissolved in chloroform (150 ml). Thischloroform solution was extracted 8 times with an aqueous saturatedsolution of NaHCO₃ (250 ml each time). The chloroform layer was thenseparated, dried (with sodium or magnesium sulfate) and evaporated underhigh vacuo (0.1 mm) at 40° (water bath) to give a light brown oil whichsolidified on cooling. This material can be used directly in the nextstep or purified as follows: The resulting oil was dissolved in 75-100ml chloroform and n-hexane (ca. 300 ml) added to precipitate out a whitecrystalline solid which was filtered from the cooled solution.(Extraction is carried out 4-8 times, until carbondioxide is no longerevolved.) This treatment was repeated two more times. Yield: 23.3 g(54%) uv λmax 267, 297 (H₂ O, pH 5.5) mp 113°-116°.

ERYTHRO-9-(2-HYDROXY-3-NONYL)6-CHLOROPURINE (VII) ##STR29##

The crude syrup from the preceding operation consisting oferythro-5-amino-4-chloro-6-(2-hydroxy-3-nonylamino)pyrimidine (11.48 g,40 mmol.) was dissolved in triethylorthoformate (106 ml) and chloroform(34 ml), ethanesulfonic acid (10 drops) was added to effect solution.After standing overnight at 25°, the solution was evaporated to a syrupunder vacuo. Yield 11.7 g (quantitative). This syrup consisting of crudeerythro-9-(2-hydroxy-3-nonyl)-6-chloropurine (VII) was used in the nextstep. λMax. 264 nm.

ERYTHRO-9-(2-HYDROXY-3-NONYL)HYPOXANTHINE (VIII)

(By hydrolysis of the 6-chloropurine derivative) ##STR30##

A suspension of erythro-6-chloro-9-(2-hydroxy-3-nonyl)purine (VII, 4.0g, 13.4 mmol) in 0.5 N NaOH (40 ml) was refluxed for 2 hr. and cooled.Neutralization with glacial acetic acid and cooling gave a crystallineprecipitate of erythro-9-(2-hydroxy-3-nonyl)hypoxanthine (VIII) whichwas filtered and dried. Yield: 3.8 g (quantitative), m.p. 196° uv λmax(pH 5.5) 251 nm.

The crude product (VIII) thus obtained was homogeneous by paperchromatography (3 solvents) and gave negative test for Cl⁻ (copper wireand flame; sodium fusion, acidification and silver nitrate).

Recrystallization of a sample of the crude material 3 times from aqueousethanol (see Purification) gave colorless crystals. m.p. 202°. Calc. forC₁₄ H₂₂ N₄ O₂ (VIII): C, 60.41; H, 7.97, N, 20.13. Found: C, 60.47; H,7.86; N, 20.08.

PURIFICATION OF ERYTHRO-9-(2-HYDROXY-3-NONYL)HYPOXANTHINE (VIII)

The crude nonyl hypoxanthine (VIII) is purified by recrystallization.The crude material is dissolved by heating in about 6-10 times itsweight in ethyl alcohol, and then an equal volume of H₂ O is added. Thesolution is treated with charcoal in an Erlenmeyer and filtered throughcelite when hot. The solution is evaporated with continuous stirring ona hot plate. Water is added in small portions to replace the evaporatedvolume until an abundant precipitate appears. Keep on evaporating thesolvent to remove all the ethyl alcohol while adding repeatedly H₂ O toreach a volume of 8-12 times the weight of material. The loss inmaterial is about 10% per each recrystallization. Two recrystallizationsraised the melting point to 202° and gave a colorless crystallineproduct while the crude material was somewhat yellow or pink and meltedat 192°.

ERYTHRO-9-(2-HYDROXY-3-NONYL)-ADENINE. HCl (IX) ##STR31##

The crude oily erythro-9-(2-hydroxy-3-nonyl)-6-chloropurine (VII) (6.15g) from the preceding preparations is dissolved in saturated methanolicammonia (300 ml) and ammonium chloride (1 g) at 80°-100° for 1 hr. in astainless steel bomb (Parr Instruments). After cooling, the solution wasevaporated to dryness in vacuo. Methanol was added and evaporated again(3 times) to eliminate the excess of ammonia.

The syrupy residue was dissolved in absolute methyl alcohol, and dry HClgas was bubbled, keeping the temperature below 20° (with an ice waterbath). After passing HCl for 1/2 hr., the mixture was cooled at 5°. Theprecipitate was collected through a sintered glass funnel, washed withcold methyl alcohol and dried in air. Yield 6.0 g (92%) m.p. 173°-175°dec. uv λmax 60 nm (in H₂ O, pH 5.5).

ALTERNATE ROUTE FOR THE PREPARATION OFERYTHRO-9-(2-HYDROXY-3-NONYL)HYPOXANTHINE (VIII) (By deamination of VII)##STR32##

Sodium nitrite (5.6 g, 71 mmole) was added slowly to a solution oferythro-9-(2-hydroxy-3-nonyl) adenine (IX, 4.0 g, 14 mmole) in 50%acetic acid (20 ml) and N HCl (3.2 ml) at 25° with stirring. The mixturewas stirred for 2 hr. at 25°. After this time, UV spectrum is monitored.When UV λmax reached 250 mm, the solution was neutralized with 2 N NaOH.The resulting precipitate was filtered and washed with H₂ O. Yield=3.03g (75%) m.p.=195°.

An analytical sample was recrystallized (3×) from water yielding aproduct m.p. 202°. Anal. Calc. for C₁₄ H₂₂ N₄ O₂ : C, 60.40; H, 7.96; N,20.13. Found: C, 60.40; H, 7.90; N, 20.12.

Method K COMPOUND NPT 15426

There was used the procedure of H. J. Schaeffer and S. F. Schwender, J.Med. Chem. 17:6 (1974.)

Method L PREPARATION OF NPT 15410

0.1 mmoles of 9-(2-hydroxy-3-nonyl)-6-hydroxy purine, NPT 15392 (27.9mg) and 0.3 mmoles of 2-hydroxypropyl, dimethylammonium4-(acetylamino)benzoate (DIP.PAcBA) (77.1 mg) were accurately weighedand dissolved in 105 ml of 0.25% sodium carbonate (NaCO₃) to yield a0.1% solution of NPT 15410 (the compound formed from NPT 15392 and(DIP.PAcBA) in a 1:3 molar ratio).

EVIDENCE FOR COMPLEX FORMATION

Phase solubility studies carried out with NPT 15392 and DIP.PAcBAdemonstrate that NPT 15392 has increased solubility at increasingconcentrations of DIP.PAcBA under conditions of constant pH. This isindicative of an interaction occuring in solution to yield a complex.

In place of the mole ratio of 1:3 (NPT 15392 and DIP.PAcBA), othercomplexes are formed by using mole ratios of 1:1 and 1:10.

Antiviral activity is shown in Tables 2 and 3.

                                      TABLE 2                                     __________________________________________________________________________     INHIBITION OF INFLUENZA VIRUS                                                REPLICATION BY 9-(HYDROXYALKYL) PURINES                                        ##STR33##                                                                    Test                 % Inhibition of Hemadsorption Foci                       Cpd Compound          Conc. (μg/ml)                                                                       Test Compound                                  No. R.sup.1                                                                           R.sup.2                                                                          X  Y       <10 10-100                                                                             >100                                           __________________________________________________________________________    15425                                                                             H   H  OH --      --  --   --                                             15428                                                                             H   H  OH DIP . PAcBA                                                                           --  --   --                                             15435                                                                             H   H  SH --          50   10                                             15437                                                                             H   H  SH DIP . PAcBA 65   65                                             15446                                                                             H   CH.sub.3                                                                         OH --       2   0    0                                             15447                                                                             H   CH.sub.3                                                                         OH DIP . PAcBA                                                                           10  26   34                                             15431                                                                             H   CH.sub.3                                                                         NH.sub.2                                                                         --          22    0                                             15432                                                                             H   CH.sub.3                                                                         NH.sub.2                                                                         DIP . PAcBA 48   62                                             15427                                                                             CH.sub.3                                                                          H  I  --                                                              15423                                                                             CH.sub.3                                                                          H  Cl --       2  13    6                                             15433                                                                             CH.sub.3                                                                          H  NH.sub.2                                                                         --          32    0                                             15434                                                                             CH.sub.3                                                                          H  NH.sub.2                                                                         DIP . PAcBA 41   62                                             15443                                                                             CH.sub.3                                                                          H  OH --           0    0                                             15444                                                                             CH.sub.3                                                                          H  OH DIP . PAcBA 44   54                                             15417                                                                             C.sub.6 H.sub.13                                                                  H  OH --      18  58   60                                             15418                                                                             C.sub.6 H.sub.13                                                                  H  OH DIP . PAcBA                                                                           16  46   52                                             15392                                                                             C.sub.6 H.sub.13                                                                  CH.sub.3                                                                         OH --      86  100  100                                            15410                                                                             C.sub.6 H.sub.13                                                                  CH.sub.3                                                                         OH DIP . PAcBA                                                                           58  96   96                                             15426                                                                             C.sub.6 H.sub.13                                                                  CH.sub.3                                                                         NH.sub.2                                                                         --      50  96   100                                            15110                                                                             --  -- -- DIP . PAcBA                                                                            0   0   20                                             __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    INHIBITION OF HERPES VIRUS REPLICATION                                        BY 9-(HYDROXYALKYL) PURINES                                                    ##STR34##                                                                                            Plaques                                                                      (PFU)                                                                         Test                                                   Compound               (15-150    Percent                                     NPT No.                                                                            R.sup.1                                                                           R.sup.2                                                                          X  Y       μg/ml)                                                                          Control                                                                             Inhibition                                  __________________________________________________________________________    15392                                                                              C.sub.6 H.sub.13                                                                  CH.sub.3                                                                         OH --                 98%                                         15417                                                                              C.sub.6 H.sub.13                                                                  CH.sub.3                                                                         OH                                                                15418                                                                              C.sub.6 H.sub.13                                                                  H  OH DIP . PAcBA                                                    15410                                                                              C.sub.6 H.sub.13                                                                  H  OH DIP . PAcBA        98%                                         __________________________________________________________________________

BIOLOGICAL ACTIVITY Methods Anti-Influenza Activity--(HemadsorptionAssay)

Upon infection of a monolayer of tissue culture cells by influenzavirus, the cell surface is altered so that guinea pig erythrocytes canbe adsorbed to the cell surface. The number of foci of adsorbed cells(hemadsorption foci forming units HAFFU) is a quantitative measure ofinfectivity. The method is as follows.

The monolayers were subcultured in the following manner: The medium waspoured off, and the monolayer washed two times with approximately 50 mlper wash of calcium and magnesium free phosphate buffered saline (PBS),(GIBCO #419) at a pH of 7.2. One ml of trypsin-EDTA solution (GIBCO#530L) containing 0.5 g trypsin (1:250) and 2.0 g EDTA/liter of ModifiedPuck's Saline A was added at 37° C. to each flask and dispersed over themonolayer with gentle shaking. The flasks were then placed in anincubator at 37° C. for approximately 3-5 minutes depending on the timerequired to dislodge the cells. Occasional shaking was required. Ten mlof planting medium was added to each flask and the cells dispersed byaspirating and expelling the suspension from the pipette. The contentsof a series of flasks were pooled and the cells in the suspension werediluted with planting medium to 7-8.5×10⁴ cells/ml. The planting mediumconsisted of the following composition: Minimum Essential Medium Eagles(MEM) with Earle's salts and HEPES buffer (GIBCO #236) supplemented byadding the following substances as specified to 87 ml of MEM:

10 ml of fetal calf serum (FCS-GIBCO #614HI)

1 ml of L-glutamine (200 Molar-GIBCO #503)

1 ml of Chlortetracycline (5000 μg/ml) GIBCO #528)

1 ml of 10,000 units penicillin, 10,000 μg streptomycin and 10,000neomycin mixture (PSN-GIBCO #564)

The cells were subcultured into Linbro tissue culture trays. The traysconsisted of 24 flat bottom wells each with a 3 ml capacity per well;the cell culture suspension (1 ml) was added to each wall.

The following day the medium was removed and replaced with freshplanting medium. The monolayers were used for experimentation when theyreached a condition in which they were almost confluent (approximately3-4 days).

When the Linbro tray HeLa cell cultures were ready for experimentation(see cells), the medium was decanted and 1 ml of maintenance medium (MEMwith FCS reduced to 3%) containing the compound being tested at a givenconcentration was added to 4 replicate cultures within a tray.

A series of different drug concentrations ranging from 2.3 to 150 μg/mlwere used. Maintenance medium alone was used for control cultures. Afterthe administration of drug and control medium, 0.1 ml of the dilutedviral suspension was added to experimental groups and infected controlcultures. Saline alone was added to non-infected control cultures. TheLinbro trays were then incubated at 37° C. for 18 hours, after whichmedia in all groups was aspirated. Each culture was washed once withPBS. The saline was aspirated and 0.5 ml of a 0.4% v/v guinea pig redblood cell suspension in PBS was added to each culture well. Thecultures remained at room temperature for 30 minutes after which themedium was decanted and culture washed 2 times with PBS to remove allbut the specifically bound red cells. After the third wash, maintenancemedium was added to all cultures.

A Howard Micrometer eyepiece (C8385) was inserted within the ocular of aNikon inverted phase contrast microscope. Each culture was scanned witha 4×low paper objective and direct counts of hemadsorbed red cells werecounted using the eyepiece grid as a field marker. Partial or completefields were counted per experimental group depending on the resultingnumber and density of hemadsorbed cells in the infected controlcultures. Magnification of 60×or 150×were chosen to obtain the bestconditions for enumerating the hemadsorbed cells. Field factors werecalculated for counting hemadsorption at 60×and 150×. At60×magnification, total field count was calculated using amultiplication factor of 55.5. At 150×magnification the multiplicationfactor was 273. The multiplication factors of 55.5 and 273 represent thetotal number of fields at 60×and 150×magnifications, respectively. Thenumber of fields counted ranged from 3 to 5 per well with 3 to 4 wellsper treatment group employed (see raw data tables in results section fornumber of fields examined). Means and standard errors were calculatedand the data was evaluated using student's t-test analysis.

BIOLOGICAL ACTIVITY Anti-Herpes Activity--(Plaque Assay)

The infection of tissue culture cells by Herpes virus causes cell lysis.After a period of time these lysed cells are visualized as a tiny cleararea (plaque) on a layer of cells. The incorporation of a test substanceinto the media will reduce the number of plaques if it is capable ofpreventing virus replication. The method is as follows:

MATERIALS AND METHODS Virus

There was employed herpes hominis type 2 purchased from American TypeCulture Collection (ATCC), Bethesda, Maryland, ATCC #VR 540, Lot 3D. Thelyophilized viral suspension was reconstituted with 1 ml steriledistilled H₂ O. The virus was passed twice through HeLa-cell monolayers.The tissue-culture supernates were pooled, dispensed in 1-ml aliquots,and stored at -70° C. The titer of this working-stock suspension wasfound to be 10⁻⁴ TCID₅₀ /0.1 ml (2 days' incubation).

Herpes Virus Plaque Assay

Vero cells in log-growth phase were subcultured at a concentration of1×10⁵ cells/ml in 50-ml Falcon flasks in Eagle's Minimum EssentialMedium (MEM), supplemented with 10% fetal calf serum (FCS) andantibiotics. Media were changed the day following planting. The Veromonolayers reached confluence by the second day after planting and withthe cells in log phase, the cultures were used for the plaque assay.

Culture media were poured off and the monolayers were wahsed once withphosphate-buffered saline (PBS). Several different dilutions of theworking-stock virus suspension were prepared and each culture flask wasinfected with 0.5 ml of one of the virus dilutions added to FCS-freemedium. This medium contained drug at a concentration of 150 μg/ml.Controls were prepared with medium devoid of drug.

Virus adsorption was allowed to proceed for 2 hours at 37° C., duringwhich time the cultures were rocked gently every 15 minutes. Then Mediawere poured off and the monolayers were washed once with 10 ml PBS.

Agarose was prepared at a concentration of 6% w/v in 50 ml PBS. A stockmedium of MEM supplemented with 2% FCS was prepared. Drug was added tosome of the stock medium at 150 μg/ml. The three solutions weremaintained at 47° C. In addition, a 1:10 dilution of pooled humananti-herpes sera was readied. Just before the start of treatment, 15 mlof the agarose solution were added to 85 ml of medium. Another 15 ml ofagarose were added to 85 ml of drug-medium.

Each of the washed monolayers in one group of experiments was treatedeither with 5 ml of agarose-medium or with 5 ml of agarose-drug-medium.In another group of experiments, each monolayer was treated either with0.2 ml of anti-herpes sera in 5 ml of stock medium, or with 0.2 ml ofanti-herpes sera in 5 ml of drug-medium. The anti-herpes sera were usedin place of agarose to localize plaques by neutralizing any free virusin the medium. The flasks were allowed to remain at room temperature for5 minutes, after which they were incubated at 37° C. for 2 days.Triplicate cultures were used for most treatment groups.

Ten ml of PBS then were added to each flask. Overlays were shaken gentlyand then were poured out of the flasks. The monolayers were stained witha solution 0.5% w/v crystal violet in 50% methanol in triple-distilledH₂ O.

Plaques were counted either directly by transmitted fluorescent lightand macroviewing, or by the use of light microscopy for microplaques.Microplaques were counted by averaging three fields per experimentalgroup under 150×magnification.

In other tests of antiviral activity the following results wereobtained:

    ______________________________________                                        % Inhibition at μg/ml                                                      Compound                                                                               0.1-1.0 1.0-10  10-100 >100  Virus                                   ______________________________________                                        15392   30-50    --      >70    >70   Influenza A                                                                   Swine 1976                                                                    (H.sub.lsw -N.sub.1)                    15417   --       50-70   >70    --    Influenza A                                                                   Swine 1976                                                                    (H.sub.lsw -N.sub.1)                    15418   --       --      >70    >70   Influenza A                                                                   Swine 1976                                                                    (H.sub.lsw -N.sub.1)                    15426   20-30    30-50   50-70   50-70                                                                              (Russian)                               15410   50-70    30-50   >70    >70   (Swine)                                 ______________________________________                                    

Additional antiviral activity tests of Compound NPT 15410 are shown inin Table 3a.

                  Table 3a                                                        ______________________________________                                        INHIBITION OF INFLUENZA VIRUS                                                 REPLICATION BY NPT 15410                                                                 Concentration Range (μg/ml)                                      Virus       .01-1.0 1.0-10.0 10.0-100                                                                             >100                                     ______________________________________                                        A/Swine/76 (H.sub.lsw N.sub.1)                                                             +++.sup.a                                                                             ++       ++++   ++++                                     A/Texas/77 (H.sub.3 N.sub.2)                                                               ++      +        ++++   ++++                                     A/Dunedin/73 (H.sub.3 N.sub.2)                                                             NT      NT       ±   ++                                       A/Jap/305 (H.sub.2 N.sub.2)                                                                NT      NT       ++++   ++++                                     A/PR.sub.8 (H.sub.0 N.sub.1)                                                               ++      ++       ++++   +++                                      A.sub.2 Hong Kong (H.sub.2 N.sub.2)                                                        NT      NT       ++     +++                                      ______________________________________                                         .sup.a NT = Not tested                                                        ± = 10-20% Inhibition                                                      + = 20-30% Inhibition                                                         ++ = 30-50% Inhibition                                                        +++ = 50-70% Inhibition                                                       ++++ = >70% Inhibition                                                   

Immunomodulation activity is shown in Table 4.

                                      Table 4                                     __________________________________________________________________________    MODULATION OF CELL MEDIATED IMMUNITY BY 9-(HYDROXYALKYL) PURINES               ##STR35##                                                                                              Maximum Percent Change                                                        Mitogen Induced (Con A)                                                                   Mitogen Induced (PHA)                                                                     Lymphokine Induced                                                            (MMF)                       Compound                  Mouse Lymph. Prolif.                                                                      Human Lymph. Prolif.                                                                      Guinea Pig Mac.                                                               Prolif.                     NPT No.                                                                             R.sup.1                                                                            R.sup.2                                                                           X   Y      .01-1.0                                                                           1.0-10                                                                            10-100                                                                            .01-1.0                                                                           1.0-10                                                                            10-100                                                                            .01-1.0                                                                           1.0-10                                                                             10-100             __________________________________________________________________________    15425 H    H   OH  --     11  0       100 0   0                               15428 H    H   OH  DIP . PAcBA                                                15435 H    H   SH  --                 55  45  -50                             15437 H    H   SH  DIP . PAcBA                                                15446 H    CH.sub.3                                                                          OH  --     6   12  0   0   0   0   0    0   0                  15447 H    CH.sub.3                                                                          OH  DIP . PAcBA                                                15431 H    CH.sub.3                                                                          NH.sub.2                                                                          --     -15 -27 -47 0   0   0                               15432 H    CH.sub.3                                                                          NH.sub.2                                                                          DIP . PAcBA                                                15427 CH.sub.3                                                                           H   I   --                                                         15423 CH.sub.3                                                                           H   CL  --                                                         15433 CH.sub.3                                                                           H   NH.sub.2                                                                          --     +15 -23 -65 53  0   -50                             15434 CH.sub.3                                                                           H   NH.sub.2                                                                          DIP . PAcBA                                                15443 CH.sub.3                                                                           H   OH  --     +17 +27 +27 0   +13 0   +20  0                      15444 CH.sub.3                                                                           H   OH  DIP . PAcBA                                                15417 C.sub.6 H.sub.13                                                                   H   OH  --     0   0   0   0   0   -50     13   -50                15418 C.sub.6 H.sub.13                                                                   H   OH  DIP . PAcBA                                                                          41  73  26              12  80   -50                15392 C.sub.6 H.sub.13                                                                   CH.sub.3                                                                          OH  --     172 162 -72 11-15                                                                             20  -50 33  23                      15410 C.sub.6 H.sub.13                                                                   CH.sub.3                                                                          OH  DIP . PAcBA                                                                          140 40  40  30-50                                                                             60      12  23                      15426 C.sub.6 H.sub.13                                                                   CH.sub.3                                                                          NH.sub.2                                                                          --     -50 -85 -91 6   -41                                 __________________________________________________________________________

Several compounds were tested for Mitogen Induced Murine LymphocyteProliferation with the following results:

    ______________________________________                                               % Stimulation at μg/ml                                              Compound 01-1.0    1.0-10    10-100  >100                                     ______________________________________                                        15392    >50%      30-50%    30-50%  not tested                               15426    0         0         0       not tested                               15410    >50       30-50     30-50   not tested                               15417    0         0         0       0                                        15418    20-30     >50       20-30   not tested                               ______________________________________                                    

BIOLOGICAL ACTIVITY Immunomodulating Assay

The following three assay procedures are used to evaluate the ability ofthe test substances to modulate the activity of several classes of cellsin the immune system. In these systems it is possible to identify bothimmunopotentiating activity (evidence by an enhancement of the parameterexamined) as well as immunosuppressant activity (evidenced by aninhibition of the parameter examined).

1. Mitogen-Induced Mouse Spleen Cell Assay

Mouse spleen cells contain a population of both B and T lymphocyteswhich can be stimulated by a number of foreign substances (e.g., plantmitogens such as Con A) to proliferate. This enhanced proliferation isan indication of enhanced cell mediated immunity. The method belowdescribes the system used to evaluate test substances asimmunopotentiators.

MATERIALS

Concanavalin A (Calbiochem, La Jolla, California), Lot #210073,lyophilized in NaCl, was prepared first as a 1% solution and diluted asa 2×concentration for each dilution (0.5, 1.0, 2.5 μg/ml).

Animals

Six to eight week old male Balb/c and C₃ H inbred mice were obtainedfrom the following sources: Flow Research Animals, Inc., Dublin,Virginia; Charles River Breeding Laboratories, Wilmington,Massachusetts; Laboratory Supply Company, Indianapolis, Indiana; andLionel Strong Foundation, San Diego, California.

Cells

Three to five mice were sacrificed by cervical dislocation and thespleens aseptically removed. Pooled spleens were minced and teased withsterile forceps; then strained through a double layer of nylon mesh. Thecell suspension was washed once with 15 ml of RPMI 1640 supplementedwith 5% fetal calf serum and antibiotics. Cells were cultured at aconcentration of 10⁶ cells/0.1 ml/well in micro-plates. Cultures wereincubated in the presence or absence of mitogen in a humidifiedatmosphere containing 5% CO₂ for 48 hours. The test compound was addedto cultures at various concentrations concommitant with mitogen.

Proliferation

Proliferation was assayed by the degree of incorporation of 1.0 μCi of[³ H] thymidine over an 18 hour incubation period. Cultures wereharvested by a MASH unit (Otto Hiller Co., Madison, Wisconsin) andthymidine incorporation was assayed by liquid scintillationspectrometry. Cultures were performed in triplicate and data areexpressed as means plus or minus the standard error of the experimentalmeans. Drug stimulation indices over control values were also calculatedand portrayed graphically.

2. Mitrogen Induced Human Peripheral Blood Lymphocytes

A clinical need exists for therapeutic agents to augment the immuneresponse in patients with deficient or depressed immune states, such asexists in viral diseases or cancer. By studying the ability of agents toaugment the proliferation of human peripheral blood lymphocytes inresponse to a foreign substance one can identify agents withimmunopotentiating activity in man. The procedure is that just set forthand that also described by Hadden et al., Infect. & Immunity, February,1976, pages 382-387, especially pages 382-383.

3. The macrophage represents a subpopulation of white blood cells whichis an important component of the immune system in control of bothcellular and humoral immunity. The assay system described belowevaluates the substances studied as potentiators of macrophage function.

Phytohemagglutinin (PHA) (HA-17) was purchased from Burroughs Wellcome.A preparation containing Macrophage Mitogen Factor (MMF) and MacrophageActivating Factor (MAF) was prepared from antigen-stimulated immunelymph node lymphocytes (guinea pig) as previously described by Hadden etal, Nature 257, 483-485 (1975). Partial purification of this preparationby vacuum dialysis and sephadex G-100 column chromatography yielded anactive fraction in the range of 35-70,000 daltons exhibiting bothmitogenic and activating properties. The active fraction was employed inboth the proliferation and activation assays.

Methods

Ficoll-hypaque purified human peripheral blood lymphocytes were preparedand PHA-induced lymphocyte proliferation was assayed by theincorporation of tritiated thymidine as described in Hadden et al.,Cell. Immunol. 20, 98-103 (1975). Each compound was analyzed in thepresence of suboptimal, optimal and supraoptimal concentrations of PHA(0.001, 0.01, 0.1 units/ml respectively). Paraffin oil-induced guineapig peritoneal macrophages were prepared and incubated as monolayerculture (>98% pure macrophages). Lymphokine (MMF)-induced proliferationwas assayed by the incorporation of tritiated thymidine at 3 and 5 daysof culture as described, Hadden et al, Nature 257, 483-485 (1975).Lymphokine (MAF)-induced macrophage activation to kill Listeriamonocytogenes following 5 days of culture in the presence or absence ofMAF was performed during a 6 hour period as described in Hadden andEngland, Immunopharmacology, pages 87-100 (Plenum Press, 1977).Phagocytosis was quantitated during a 20-minute exposure to Listeriamonocytogenes by counting the number of macrophages containing bacteriaand the number of bacteria per phagocytic cell on gram stainedmonolayers in Labtek chambers. Intracellular killing of bacteria wasevaluated by counting the number of cells containing bacteria and thenumber of bacteria/cell 6 hours after the initial 20 minute exposure.Parallel experiments in which macrophages were lysed and intracellularbacteria were cultured confirm the validity of bacterial activitydetermined by this manner in this system. The drugs were employed ineach of the three systems over serial log concentration range intriplicate in the presence and absence of mitogen or lymphokine. Eachtype of experiment was performed at least three times. Previousexperiments indicate a parallelism of response to pharmacologicmodulation in the proliferation and achivation assays.

BIOLOGICAL ACTIVITY Anti-Leukemic Activity (Inhibition of L-1210 Growth)

Leukemic cells isolated from mice bearing the L-1210 tumor are culturedin vitro and their growth can be measured by counting the number ofcells in the culture over a period of time. The incorporation of a testsubstance into the media will prevent the growth of the leukemic cells,an indication of an effective anti-leukemic agent.

I₅₀ (concentration of drug inhibiting growth of L-1210) by % for thetested compounds was as follows:

    ______________________________________                                                           Concentration                                              Compound           (micrograms/ml)                                            ______________________________________                                        15392              28                                                         15410              54                                                         15417              47                                                         15418              70                                                         ______________________________________                                    

The assay system used is set forth below.

To Measure Inhibition of Leukemic Cell (L-1210) Growth

Check to see that there is adequate cell growth in the stock cultures.Use cells 48-72 hours after transfers are done.

Weigh out the drugs at 50 times the desired final concentration and madeserial dilutions.

Make up the final medium using 500 mls McCoy's 5A medium, 15% fetal calfserum, 5 mls penicillin-streptomycin solution, and 5 mlsantibiotic-antimycotic solution and let it stand at room temperature.

Using sterile technique, add 0.1 ml of the drug dilutions to each tube.

Add an appropriate quantity of cells to the prepared medium. Aftermixing, remove a 0.5 ml sample, place it in a vial containing 9.5 mls ofsaline, and count it on the Coulter Counter. Multiply the count by 40 tocompensate for the 40 fold dilution (0.5 ml into 0.5 ml saline andrecord the inoculum.

Add 5 mls of cell suspension to each tube. Swirl the bottle every 4tubes to insure a more uniform distribution of cells.

Tighten the caps and place in the CO₂ incubator at 36°-38° for 96 hours.

After 96 hours remove the tubes from the incubator and count thecontents of each on the Coulter Counter. Multiply all counts by 40 andaverage the four counts for each drug dilution. If the count is lessthan the inoculum, record 100% inhibition. If the count is greater thanthe average of the eight control counts, record 0% inhibition. For allother counts use the following formula: ##EQU1## 100%-%servival=inhibition of growth due to treatment.

The subject compounds of this invention have been shown to inhibit thereplication of a representative sample of both RNA and DNA viruses usingstandard tissue culture techniques. In the case of the RNA viruses,several strains of influenza virus belonging to both the A and Bsub-types were shown to be inhibited, using the hemadsorption technique(Section II, B). The specific compounds found to inhibit influenza virusreplication (Type A/USSR 90) are shown in Table 1. Several members ofthe Series NPT 15392, NPT 15410, NPT 15417, and NPT 15418 were shown toinhibit the replication of at least 4 different strains of influenzavirus at concentrations ranging from 1-150 μg/ml.

In addition, several members of the Series, NPT 15410 and 15392, havebeen shown to inhibit the replication of Herpes Simplex virus, a memberof the DNA class of viruses and a virus responsible for severemucocutaneous lesions in man, along with the fatal Herpes encephalitis.Other members of this class of viruses are responsible for hoof andmouth disease in swine and cattle and infectious rhinotracheitis in catsand kennel cough in dogs. Even concentrations less than 100 μg/ml of NPT15392 and 15410 were found to reduce plaque formation caused by HerpesSimplex virus to an extent of >90%. Other members of the RNA and DNAclass of viruses are shown in Table 5 and are responsible for thediseases specified. Of all the diseases in the world at least 25% areknown to be caused by viruses. In addition, a number of viruses havebeen isolated that are shown to produce tumors. Thus, antiviral agentsmay be expected to, by themselves, have some antitumor properties.

It is an established fact that many infectious agents, such as viruses(influenza virus, HSV, Friend leukemia virus), bacteria and fungi causean immune suppressed stated in the host, weakening his defenses toinfection by infectious agents. Most other antiviral antimetabolitesubstances, like AraC, cause a suppression of host immune defensemechanisms, thereby exhibiting potential to lessen the body's ownnatural defense mechanisms and enhance secondary infection.

An immunopotentiator or immunomodulator is any agent which eitherrestores depressed immune function, or enhances normal immune function,or both. Immune function is defined as the development and expression ofhumoral (antibody-mediated) immunity, cellular (thymocyte-mediated)immunity, or macrophage and granulocyte mediated resistance. Itlogically includes agents acting directly on the cells involved in theexpression of immune response, or on cellular or molecular mechanismswhich, in turn, act to modify the function of cells involved in immuneresponse. Augmentation of immune function may result from the action ofan agent to abrogate suppressive mechanisms derived by negative-feedbackinfluences endogenous or exogenous to the immune system. Thus, immunepotentiators have diverse mechanisms of action. Despite the diversity ofcell site of action and biochemical mechanism of action ofimmunopotentiators, their applications are essentially the same; thatis, to enhance host resistance.

Applications of Immunopotentiators

(1) The principal protective function of the immune system relates toresistance to invasion by pathogens, including viruses, rickettsia,mycoplasma, bacteria, fungi, and parasites of all types. Thus,improvement of immune response, particularly when depressed, wouldcalculatedly improve resistance in infection or infestation by any ofthe above pathogens. An immunopotentiator alone or in combination withanti-infective therapy can be applied to any and all infectiousdiseases.

(2) A second protective function of the immune system is thought to beresistance to engraftment of foreign tissue, either natural as in thefetal-maternal relationship; or unnatural as performed by the transplantphysician. Immunopotentiators can also be used to facilitate rejectionof fetal or placental tissues or to modify or induce tolerance tografts.

(3) A third protective function of the immune system is thought to beresistance to malignant cell development as in cancer. The use ofimmunopotentiators can be used in cancer treatment to enhance tumorrejection and to inhibit tumor recurrences following other forms oftherapy.

(4) A fourth protective function involves the capacity to recognizeforeign-ness and to maintain non-reactivity to self by positivesuppressor mechanisms. In auto-immune and related disorders, immunereactivity directed at self antigens or exaggerated, elevated responsesare apparent which are self-destructive. Immunopotentiators can be usedto restore normal suppressor mechanisms, induce tolerance, or otherwisepromote a normal immune response.

Each of the protective functions of the immune system can be modified bynon-specific therapy with immunopotentiators alone or in combinationwith other agents employed to improve resistance or to kill the invadingpathogen. In addition, specific resistance can be augmented by use ofimmunopotentiators in conjunction with some form of antigen as in avaccine employing, for example, virus, tumor cell, etc. This use can beto induce either specific immunity or tolerance. The latter might beexemplified by use with antigen in allergy or auto-immune diseases. Useof immunopotentiators may be either therapeutic or prophylactic; thelatter particularly in aging, where infection, auto-immunity, and cancerare more common. The timing of administration and routes are variableand may be critical indetermining whether a positive or negativeresponse results. Any agent capable of augmenting immune response mayinhibit it depending on timing and dose; thus, under certaincircumstances an immunopotentiator could be used as an immunosuppressiveagent for use in allergy, auto-immunity and transplantation.

Table 4 above presents the results of an evaluation of a number of thesesubject compounds as potentiators of the immune response. Threedifferent test systems were used. The first involves a measure of theability of the test compound to enhance the ability of mouse lymphocytesto proliferate in response to a plant mitogen (Con A). The secondinvolves measuring the ability of the test compounds to enhance humanlymphocyte proliferation in response to a second plant mitogen (PHA).The third system measures the ability of these test substances toenhance macrophage proliferation in response to a natural lymphokine(MMF, Macrophage Mitogenic Factor). This latter response, theproliferation and activation of macrophages, has been shown to beinvolved in the killing of bacteria, viruses and tumor cells by thisclass of white blood cells.

Specificant potentiation of the immune response has been observed by15392, 15410, and 15418.

Finally, the activity of several of these agents, NPT 15392 and 15410 asinhibitors of the growth of abnormal lymphocytes has been determined.Notably, both substances are capable of inhibiting the proliferation ofmouse leukemic lymphocytes (an L-1210 cell line) in tissue culture. A50% inhibition of L-1210 cells was effected by NPT 15392 at 28 μg/ml andby NPT 15410 at 54 μg/ml. The ability to inhibit leukemic lymphocytes atconcentrations that stimulate normal lymphocytes is a unique propertynot known to be present in any other class of substances.

The products of the present invention are members of a class ofsubstances, which specifically inhibit the replication of RNA and DNAvirus, modulate (potentiate) the immune response and inhibit the growthof leukemic lymphocytes. Based on in vitro experiments, whichdemonstrate activity over a concentration range of 0.01-150 μg/ml, doseranges effective in mammals are 0.05-500 mg/kg. A lack of toxicity hasbeen noted at levels of 1,500 mg/kg in mice for certain numbers of thisseries.

The immunopotentiators of the invention can be employed, for example, toprovide resistance to invasion by the viruses in Table 5.

                  Table 5                                                         ______________________________________                                        Virus            Class   Disease                                              ______________________________________                                        Arenavirus       RNA     Rift Valley Fever                                    Influenza        RNA     Influenza                                            Rhinovirus       RNA     Common Cold                                          Poliovirus       RNA     Polio                                                Measles          RNA     Rubella                                              Newcastles Disease Virus                                                                       RNA     Newcastles disease                                   Rotavirus        RNA     Gastroenteritis in infants                           Hepatitis Type A RNA     Infectious Hepatitis                                 Rabies virus     RNA     Rabies                                               Arbovirus        RNA     Encephalitis                                         Vaccinia virus   DNA     Smallpox                                             Herpes Simplex Virus                                                                           DNA     Cold sore, Encephalitis,                                                      Venereal Disease                                     Herpes Zoster    DNA     Shingles                                             Varicella Zoster DNA     Chicken pox                                          Adenovirus       DNA     Respiratory                                          Hepatitus Type B DNA     Chronic Hepatitis,                                                            Severe Hepatitis                                     Hoof and Mouth Disease virus                                                                   DNA     Hoof and Mouth Disease                               Machupo Virus            Hemorrhagic Fever                                    ______________________________________                                    

POTENTIATION BY DIP.PAcBA OF BIOLOGICAL ACTIVITIES

Of the substances described in Table 1, NPT 15392 and NPT 15446 are newcompounds claimed in the application of Alfredo Giner-Sorolla filed oneven date. Also new are the DIP.PAcBA salts presented in this table,namely, 15428, 15437, 15447, 15432, 15434, 15444, 15418 and 15410. NPT15392, NPT 15417, NPT 15426 have all been shown to have significantanti-influenza activity by themselves. In one instance (with NPT 15392)the addition of DIP.PAcBA salt to NPT 15392 to form 15410 does notpotentiate the anti-influenza activity. In the case of NPT 15417,addition of DIP.PAcBA salt to form 15418 does potentiate theanti-influenza activity. A summary of the relative ability of DIP.PAcBAsalts to potentiate the different biological activities is set forthbelow.

                  Table 6                                                         ______________________________________                                                                              Immuno-                                 Com-  Dip . PAcBA                                                                              Anti-      Potentiation                                                                            potent-                                 pound Salt       Influenza  Anti-Leukemia                                                                           iation                                  ______________________________________                                        15392 15410      Both are   Yes       Yes                                                      equally active                                               15417 15418      Yes        --        Yes                                     15435 15437      Yes        --        --                                      15446 15447      Yes        --        --                                      15431 15432      Yes        --        --                                      15433 15434      Yes        --        --                                      15443 15444      Yes        --        --                                      ______________________________________                                    

FORMULATIONS

The compounds of the present invention can be fed to a mammal at adosage of 1-1000 mg/kg of body weight and are believed to be active atlevels as low as 0.05 mg/kg. The LD₅₀ as determined in mice of NPT 15410given intraparenterally was 4,300 mg/kg, while subcutaneously was 4,900mg/kg. NPT 15392 has been given to mice at doses of 1000 mg/kg and nodrug related mortality was noted.

They can be administered in tablet or capsule form to humans and wheresolubility permits in the form of syrups or injectable solutions orwhere insoluble as suspensions. Typical pharmaceutical formulations aredescribed below:

Capsule:

NPT 15392: 50-500 mg.

Avicel pH 101 (microcrystalline cellulose): to make 800 mg.

Suspension:

Aqueous suspensions can be made with a number of suspending agentsincorporated with the active drug substances. Included as suspendingagents are such substances as sodium carboxymethylcellulose, Naalginate, gum tragacanth, Avicel RC-591 (microcellulose),methylcellulose, Veegum, Xanthan gum. In addition to a suspending agentsuch substances as sweeteners, flavors, colorants, preservatives,protective colloids and dispersants may be added.

    ______________________________________                                        TABLET FORMULATION                                                            ______________________________________                                        NPT 15392              50-500 mg                                              Avicel pH 101          130 mg                                                 Starch, modified       20 mg                                                  Magnesium stearate U.S.P.                                                                            5.5 mg                                                 Polyvinylpyrrolidone   22 mg                                                  Stearic acid U.S.P.    30 mg                                                  ______________________________________                                    

    ______________________________________                                        SYRUP FORMULATION                                                             ______________________________________                                        NPT 15392        25-125 mg (or at maximum                                                                level of                                                                      solubility)                                        Corn Sugar       3.25 g.                                                      Distilled Water  .05 g.                                                       FD and C Red 40  .00175 g.                                                    Sodium Saccharin .00250 g.                                                    Alcohol U.S.P.   .08 g.                                                       Methyl paraben U.S.P.                                                                          .005 g.                                                      Propyl paraben U.S.P.                                                                          .001 g.                                                      Glycerin         .31225 g.                                                    Cherry flavor    .00825 g.                                                    Fruit flavor     .00825 g.                                                    Distilled water g.s.ad                                                                         5 ml.                                                        ______________________________________                                    

IN VIVO TREATMENT OF MICE WITH NPT 15392 AND NPT 15410: EFFECT ON THE INVITRO STIMULATION OF SPLEEN CELL PROLIFERATION BY CONCANAVALIN A

The purpose of this study was to determine the effects of in vivotreatment of mice with the compounds NPT 15392 and 15410 on thesubsequent activity of spleen cells isolated from these animals andevaluated in vitro for their proliferative response to the mitogen,Concanavalin A (Con A).

PROCEDURE In Vivo Treatment

Nine male Balb/C mice, 8-9 weeks old, weighing 18-20 gms were dividedinto three groups. One group was treated twice daily (for 1 day), in themorning and afternoon, with an oral dose of NPT 15392 at 10 mg/kg. Thesecond group was similarly treated with NPT 15410 at 20 mg/kg. A thirdgroup, dosed with saline served as a placebo control.

In Vitro Spleen Cell Assay: Cell Preparation

The following day, each group was sacrificed and the spleens removed andpooled. The spleens were minced and the cells washed in RPMI-1640 medium(Grand Island Biologicals) supplemental with 2 mm glutamine andantibiotics. The cell concentration of each preparation was determinedby a Coulter counter and adjusted to 5×10⁶ cells/ml with RPMI medium.

Microtiter Plate Assay

Microtiter assays were carried out in 0.2 ml incubations, containing5×10⁵ cells and Con A or Con A and compounds at the indicatedconcentrations. All assays were performed with 6 replicates and comparedwith a blank assay containing only cells. The assay plates wereincubated at 37° in 5% CO₂ for 4 days. During the final 18-20 hours ofincubation, 0.5 ml of ³ HTdR (10 μCi/ml, 6 C_(i) /m mole) were added toeach culture. The cultures were harvested with a multiple automaticsample harvester (MASH) unit and the incorporated ³ HTdR determined witha Beckman LS 8000 liquid scintillation counter, as a measure of cellproliferation. The results are tabulated as the ratio of the activity inthe Con A or Con A and compound treated cultures to the blank cultures.

In vivo treatment with either compound 15392 or 15410 increases thesubsequent response of the spleen cells, in vitro, to Con A stimulationat a suboptimal mitogen concentration (5 μg/ml. Thus compound 15410increased the stimulation ratio to 100:1 compared to 55:1 with theplacebo. No significant differences are obtained with either compound15392 or 15410 treatment when the cells are stimulated with a moreoptimal concentration of Con A (10 μg/ml).

There was also tested the effect of subsequent in vitro treatment of ConA stimulated cells with NPT 15392 and 15410 at 1 μg/ml. Both compoundsshow a marked ability to augment the Con A stimulation, particularly atthe suboptimal mitogen conecntration (5 μg/ml) and to a lesser extent at10 μg/ml. At 5 μg/l of Con A, the stimulation by NPT 15392 is 2.8 foldover Con A alone, while that for NPT 15410 is 3.3 fold.

These results indicate an immunomodulating effect of these compounds onspleen cell proliferation. Pre-treatment of animals with eithercompounds with sensitize the cells to subsequent mitogenic stimulationwhile exposure of the cells in vitro to the compounds followingmitogenic stimulation will augment the proliferative responseparticularly under conditions when the response to mitogen alone is low.

What is claimed is:
 1. A method of imparting immunomodulating activity,antiviral activity or anti-leukemic activity comprising administering toa mammal an effective amount for such purpose of a compound of theformula ##STR36## where X is OH, NH₂, SH, OR or SR (where R is alkyl of1 to 4 carbon atoms or benzyl), R¹ is H or alkyl of 1 to 8 carbon atoms,R² is H or methyl, Y is the salt of an amine of the formula ##STR37##where R³ and R⁴ are lower alkyl, n is an integer from 2 to 4 and where zis 0 or a number from 1 to 10 with a pharmaceutically acceptable acid.2. A method according to claim 1 wherein when z is 1 to 10 the acid isp-acetamidobenzoic acid.
 3. A method according to claim 2 wherein z is 1to
 10. 4. A method according to claim 3 where R¹ is H or n-alkyl of 1 to8 carbon atoms and R³ and R⁴ are alkyl of 1 to 4 carbon atoms.
 5. Amethod according to claim 4 where R¹ is H or n-alkyl of 1 to 7 carbonatoms.
 6. A method according to claim 5 where R¹ is H or n-alkyl of 1 to6 carbon atoms.
 7. A method according to claim 6 where R¹ is n-hexyl. 8.A method according to claim 3 where X is OH, NH₂ or SH.
 9. A methodaccording to claim 8 where X is OH.
 10. A method according to claim 8where X is NH₂.
 11. A method according to claim 8 where X is SH.
 12. Amethod according to claim 3 where R² is H.
 13. A method according toclaim 12 where X is OH.
 14. A method according to claim 12 were X isNH₂.
 15. A method according to claim 12 where X is SH.
 16. A methodaccording to claim 3 where R² is methyl.
 17. A method according to claim16 where X is OH.
 18. A method according to claim 16 where X is NH₂. 19.A method according to claim 16 where X is SH.
 20. A method according toclaim 4 where X is OH, NH₂ or SH and Y is the salt ofdimethylaminoisopropanol and p-acetamidobenzoic acid.
 21. A methodaccording to claim 20 where X is OH.
 22. A method according to claim 20where R² is hydrogen.
 23. A method according to claim 20 where R¹ isn-hexyl.
 24. A method according to claim 20 where R¹ is methyl.
 25. Amethod according to claim 20 where R² is methyl.
 26. A method accordingto claim 25 where R¹ is n-hexyl.
 27. A method according to claim 26where R¹ is methyl.
 28. A method according to claim 3 where X is OH, NH₂or SH and R¹ is hydrogen.
 29. A method according to claim 3 wherein X isOH, R¹ is n-hexyl and R² is methyl.
 30. A method according to claim 29wherein z is
 3. 31. A method according to claim 30 where Y is the saltof dimethylaminoisopropanol with p-acetamidobenzoic acid.
 32. A methodaccording to claim 29 where Y is the salt of dimethylaminoisopropanolwith p-acetamidobenzoic acid.
 33. A method according to claim 3 where R²is H.
 34. A method according to claim 3 where X is NH₂ and the compoundis used in an amount to impart immunoinhibiting activity.
 35. A methodaccording to claim 3 where R² is methyl.
 36. A method according to claim3 wherein the compound is used in an amount to impart immuno-modulatingactivity.
 37. A method according to claim 36 wherein the compound isused in an amount to impart immuno-stimulating activity.
 38. A methodaccording to claim 37 where X is OH.
 39. A method according to claim 3wherein the compound is used in an amount to impart antiviral activity.40. A method according to claim 39 where X is OH.
 41. A method accordingto claim 3 wherein the compound is used in an amount to impartantileukemic activity, X is OH, R¹ is alkyl of 1 to 8 carbon atoms andR² is methyl.
 42. A method according to claim 41 where R¹ is n-hexyl.43. A method according to claim 42 where Y is the salt ofdimethylaminoisopropanol with p-acetamidobenzoic acid.
 44. A methodaccording to claim 43 wherein the compound is administered to a mammalhaving leukemia.
 45. A method according to claim 36 wherein the compoundis used in an amount to impart immunoinhibiting activity.
 46. A methodaccording to claim 45 wherein X is OH.
 47. A method according to claim46 where R¹ is n-hexyl.
 48. A method according to claim 47 where R² ismethyl.
 49. A method according to claim 1 where z is
 0. 50. A methodaccording to claim 49 where R¹ is H or n-alkyl of 1 to 8 carbon atomsand R³ and R⁴ are alkyl of 1 to 4 carbon atoms.
 51. A method accordingto claim 50 where R¹ is H or n-alkyl of 1 to 7 carbon atoms.
 52. Amethod according to claim 51 where R¹ is H or n-alkyl of 1 to 6 carbonatoms.
 53. A method according to claim 52 where R¹ is n-hexyl.
 54. Amethod according to claim 49 where X is OH, NH₂ or SH.
 55. A methodaccording to claim 54 where X is OH.
 56. A method according to claim 54where X is NH₂.
 57. A method according to claim 54 where X is SH.
 58. Amethod according to claim 54 where R² is H.
 59. A method according toclaim 58 where X is OH.
 60. A method according to claim 58 where X isNH₂.
 61. A method according to claim 58 where X is SH.
 62. A methodaccording to claim 54 where R² is methyl.
 63. A method according toclaim 62 where X is OH.
 64. A method according to claim 62 where X isNH₂.
 65. A method according to claim 62 where X is SH.
 66. A methodaccording to claim 49 where X is OH, NH₂ or SH and R¹ is hydrogen.
 67. Amethod according to claim 66 where X is OH.
 68. A method according toclaim 49 where X is OH, R¹ is n-hexyl and R² is methyl.
 69. A methodaccording to claim 49 wherein the compound is used in an amount toimpart immunomodulating activity.
 70. A method according to claim 69wherein X is NH₂ and the compound is used in an amount to impartimmunoinhibitory activity.
 71. A method according to claim 69 whereinthe compound is used in an amount to impart immunostimulating activity.72. A method according to claim 71 where X is OH.
 73. A method accordingto claim 72 where R² is methyl.
 74. A method according to claim 73 whereR¹ is 1 to 6 carbon atoms n-alkyl.
 75. A method according to claim 74where R¹ is n-hexyl.
 76. A method according to claim 69 wherein thecompound is used in an amount to impart immunoinhibiting activity.
 77. Amethod according to claim 76 where X is OH.
 78. A method according toclaim 77 where R¹ is n-hexyl.
 79. a method according to claim 76 whereR² is methyl.
 80. A method according to claim 49 wherein the compound isused in an amount to impart antiviral activity.
 81. A method accordingto claim 80 where X is OH.
 82. A method according to claim 81 where R²is methyl.
 83. A method according to claim 82 where R¹ is n-hexyl.
 84. Amethod according to claim 49 wherein the compound is used in an amountto impart anti-leukemic activity, X is OH, R¹ is alkyl of 1 to 8 carbonatoms and R² is methyl.
 85. A method according to claim 84 where R¹ isn-hexyl.
 86. A method according to claim 85 wherein the compound isadministered to a mammal having leukemia.
 87. A method according toclaim 1 wherein when the activity is antiviral the virus is influenzavirus or herpes virus.
 88. A method according to claim 39 wherein thevirus is influenza virus or herpes virus.
 89. A method according toclaim 88 where X is OH.
 90. A method according to claim 80 wherein thevirus is influenza virus or herpes virus.
 91. A method according toclaim 90 wherein X is OH.
 92. A method according to claim 91 wherein R²is methyl.
 93. A method according to claim 92 wherein R¹ is n-hexyl.